Summary Non-coding RNAs (ncRNA) represent the majority of the human transcriptome. The long non-coding RNAs (LncRNA) regulate a myriad of cellular, developmental, immune response and disease-associated processes and are emerging as diagnostic biomarkers. Recent reports have demonstrated the importance of cellular LncRNAs on HIV replication and latency. LncRNAs interact with HIV proteins but the precise roles of these interactions in viral replication, latency, and reactivation are largely unknown. LncRNAs have been shown to swiftly and efficiently regulate cellular trafficking, localization, and function of their protein binding partners. LncRNAs are modulated upon viral infection, thus they can rapidly interact with viral proteins and influence viral replication cycle in the cells. However, the possibility that the cellular LncRNAs interact with viral proteins directly to enhance or interfere with their function and affect viral replication has not been explored systematically. The HIV-Tat protein is known to interact with cellular RNAs and at least one LncRNA, NRON. In our preliminary investigation, we found 20 novel interactions between Tat and cellular LncRNAs. One of the Tat binding LncRNAs, metastasis associated lung adenocarcinoma transcript 1 (MALAT1) inhibited HIV replication in a cell line model as well as ex vivo infected primary CD4+ T cells. We hypothesize that MALAT1 influences HIV infection through its interaction with Tat. In this proposal, we seek to develop a Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) loss-of-function screen and determine the effects of top 5 Tat-binding cellular LncRNAs on HIV replication (Aim1) and molecular mechanisms of MALAT1 mediated HIV inhibition (Aim2). The proposed study will be the first to comprehensively dissect the impact of Tat binding cellular LncRNAs on HIV. The LncRNAs found to affect HIV replication can be used as potential therapeutic targets. The data generated will expand the current information on the role of cellular LncRNAs in HIV infection, discover functionally relevant novel interactions between LncRNAs and HIV proteins and identify molecular targets for anti-HIV therapy.